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TCA
4-8 days very slow actingGood nuclear stainingWeak
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Sulfurous
WeakFor minute pieces of bone
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Chromic Acid (Flemmings fluid)
Chromic acid, osmium tetroxide, glacialHAc
Fixative and decalcifier Nuclear staining with hematoxylin isinhibitedForms precipitate at the bottom*carcinogenic, corrosive to skin
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Citric Acid citrate buffer pH4.5
With zinc sulfate and chloroform6 days slowGood nuclear and cytoplasmic staining
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HNO3 - summary
Most common, rapid (1-3 days)Yellow color imparted prevented by 5%
sodium sulfate, wash in running water x 12hours or + 0.1% ureaFormol Nitric (1-3 days)Perenyis w/ chromic acid + abs. EtOH
Decalcifies & softens; slow (2-7 days)
Phloroglucin Nitric most rapid (12-24h); poor nuclear staining
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HCl slower than HNO3
Formic fixative/decalcifier, slowFormic Acid-Sodium Citrate solution - slow
TCA good nuclear staining, no washingout; weak & slow actingSulfurous very weakChromic (Flemmings) fixative/decalcifier Citric Acid Citrate buffer pH4.5 - slowVon Ebners NaCl, HCl
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Chelating Agents
EDTA (versene)Excellent stainingVery slowSlight tissue hardening produced
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Ion Exchange Resins
* artifacts produced, usually caused byCO2 bubbles
slow
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Electrophoresis(Electrical ionization)
Formic acid, conc HCl, distilled water
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Tests for Completeness of Decal:
Physical / MechanicalX ray / RadiologicalChemical litmus paper red if due toacidity, add NH3 drop by drop bluelitumus; if cloudy still w/ calcium; if clear:+ammonium oxalate, 30 mins cloudy if incomplete
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Tissue softeners
Perenyis 12 24 hours4% aqueous phenol 1 3 daysMolliflex (swollen & soapy appearance)2 % HCl1 % HCl in 70 % alcohol
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*Post decalcification
Remove acid by saturated lithiumcarbonate solution or 5-10 % aqueous
NaHCO3 for several hoursRunning tap water If EDTA is used use 70 % alcohol
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*Rate of decalcification
More concentrated acid solutions morerapid but more harmful to tissue
Heat hastens decalcification, butincreases damaging effect of acids totissues
Mechanical agitation, sonicationIdeal time 24 to 48 hours
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Dehydration most critical stageof processing difficult to correct
Ideal Dehydrating Solution:Rapid acting w/ no tissue shrinkage or
distortionShould not evaporate fastCan dehydrate even fatty tissues
Non toxicNot fire hazardShould not harden tissues excessively
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Dehydrating Agents:
Alcohol most common AcetoneDioxaneCellosolveTriethyl phosphateTHF
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Alcohol progressively increasing
Ethyl best, fast, mixes with water andpenetrates easily, not poisonous, cheap
Methyl toxic, for blood filmsButyl slow, for Plant and Animalmicrotechnique
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* Alcohol
Prolonged storage in 70 % alcohol macerates tissues
Directly placed in high grade alcohol shrinkage & hardening of tissues
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Acetone
CheapRapid actingVolatileTissue shrinkage
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Dioxane (Diethylene Dioxide)
Excellent dehydrating & clearing agentLess tissue shrinkage
Tissues can be left here for a long timeRibbons poorlyExpensive
Dangerous vapor cumulativeGraupners method (dioxane, paraffin wax) Weisebergers method (gauze, Ca oxide)
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Cellosolve
Ethylene Glycol Monoethyl Ether RapidStorage without producing hardening or distortion
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Triethyl Phosphate
Minimum shrinkageMinimum distortion and hardening of tissue
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Tetrahydrofuran - THF
Dehydrates and clearsLess shrinkageEasier cutting w/ fewer artifactsNon toxic; 6 months exposure conjunctival irritationOffensive odor (use well ventilated room)
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*additives to dehydrating agents
4 % phenol added to 95 % ethanol softens hard tissues
Hard tissues immerse in glycerol/alcoholmixture or in Molliflex
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Clearing / Dealcoholization
Substance will dissolve wax with which thetissue is to be impregnated
After staining transparent tissuesHigh refractive index
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Clearing Agents:
Miscible with alcohol and paraffin &/or mounting medium
Should not produce excessive tissueshrinkage & hardeningShould not evaporate quickly in a
waterbathShould make tissues transparent
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Clearing Agents:
XyleneToluene
BenzeneChloroformCedarwood oil
Aniline oilClove oilCCl4
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* Clearing Agents (cont)
* methyl benzoate and methyl salicylateslow
`THFDehydrates and clearsNon toxic
Offensive odor
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Xylene (Xylol)
Most common, colorlessMost rapidCheapMilky if incomplete dehydrationHardens/shrinks tissues not for nervoussystem & lymph nodesHard/brittle tissues if > 3 hours
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Toluene
Not carcinogenic; but will emit fumes thatare toxic upon prolonged exposure
Rapid actingTissues do not become excessively hardand brittle even if left here for 24 hours
Slower than xylene & benzeneexpensive
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Benzene
Rapid actingVolatilizes rapidly in paraffin ovenTissue shrinkage if left for a long timeCarcinogenic; aplastic anemia